An induction method for Arundo donax polyploid

Abstract

The present invention provides a method for inducing Arundo donax polyploids. Through the induction method of the present invention, on the basis of rapid propagation of Arundo donax by tissue culture, the culture medium can be scientifically formulated, the culture conditions can be reasonably adjusted, and Arundo donax polyploids with excellent traits can be obtained quickly and efficiently, effectively reducing the incidence of chimeras. The present invention uses the tillering tissue mass of clustered buds as the induction material, which enriches the existing Arundo donax polyploid induction materials compared with the traditional Arundo donax polyploid induction materials. By using colchicine at a specific concentration to induce the tillering tissue mass, the induction rate of Arundo donax polyploids is increased, the incidence of chimeras is effectively reduced, time and costs are saved, and a new way is provided for the breeding of new Arundo donax varieties.

Description

Technical Field

[0001] The present invention relates to the technical field of plant breeding, and particularly relates to a method for inducing polyploid Arundo donax. Background Art

[0002] Arundo donax is a perennial plant of the Poaceae family and Arundo genus. It has a well-developed rhizome, thick and upright, tough culms with many nodes and often branched. It can be used in various industries such as feed, ethanol, papermaking, power generation, and environmental remediation. The existing methods for propagating Arundo donax include sowing, dividing plants, cutting, etc. Generally, the method of dividing plants is mainly used. Usually, in early spring, it is cut into clusters with 4 - 5 buds along the periphery of the plant and then transplanted. This method has a slow propagation speed, the seedlings are easily virus-infected, and it has a high labor intensity, inconvenient transportation, and high afforestation costs. It is difficult to solve the problem of short supply of seedlings in a short time and is not conducive to the industrial production of Arundo donax.

[0003] Compared with traditional methods, using tissue culture technology to propagate Arundo donax can obtain a large number of uniform virus-free seedlings in a short time, and the growth advantages of the seedlings are obvious. It is an effective way for the industrialized breeding of Arundo donax seedlings. Polyploid breeding is an important means for cultivating new plant varieties. Polyploid breeding originated in the early 20th century and is now widely used in the cultivation of new plant varieties. Due to the increase in genome ploidy, polyploids often show characteristics such as gigantism in morphology and enhanced resistance. However, traditional polyploid breeding methods mainly use germinating seeds, callus, seedlings, the growing points of young roots or stems, and germinating buds of bulbs or corms as induction materials. The cell physiological states of these materials vary greatly, and chimeras are more likely to appear during polyploid induction, and the traits are unstable. Summary of the Invention

[0004] In view of this, the present invention proposes a method for inducing polyploid Arundo donax.

[0005] The technical solution of the present invention is realized as follows:

[0006] A method for inducing polyploid Arundo donax, comprising the following steps:

[0007] S1. Collect strong stem segments of Arundo donax, remove bracts, select stem segments with buds, wash and disinfect to obtain pretreated explants;

[0008] S2. Inoculate the pretreated explants in step S1 on an MS solid medium and culture under light to obtain sterile buds;

[0009] S3. Inoculate the sterile buds in step S2 on medium B and culture under light to obtain cluster buds;

[0010] S4. Immerse only the tillering tissue mass of the cluster buds in step S3 in liquid medium C to obtain the tillering tissue mass after polyploid induction;

[0011] S5. Inoculate the tillering tissue mass after polyploid induction in S4 onto liquid medium D for culture to obtain regenerated plants;

[0012] S6. Identify the ploidy of the leaves of the regenerated plants in S5 by flow cytometry;

[0013] S7. Plant and acclimatize the regenerated plants in S6. When the regenerated plants grow to more than 20 cm, take the leaves for re-identification.

[0014] Further, the specific step S1 is as follows: Under the condition that new bamboo shoots of Arundo donax grow, new leaves at the bottom grow but the top bracts do not fall off and the leaves do not emerge, collect strong stem segments of Arundo donax, remove the bracts, cut them into 2 - 3 cm stem segments with buds, wash them, soak them in 70% v / v ethanol solution for 30 - 60 s in a sterile environment, rinse them with sterile water for 1 - 3 times, then soak them in 0.1% w / v mercuric chloride solution for 10 - 12 min, and rinse them with sterile water for 5 - 7 times to obtain pretreated explants.

[0015] Further, the specific step S2 is as follows: Under sterile conditions, cut off 2 - 3 mm from each end of the stem segment of the pretreated explant in S1, inoculate it on MS solid medium, make the bud point contact the surface of the medium, and culture it at a temperature of 24 - 27 °C and a light time of 12 h / 24 h for 15 - 30 d to obtain sterile buds.

[0016] Further, the specific step S3 is as follows: Cut the sterile buds in S2 into stem segments with buds, inoculate them on medium B, and culture them at a temperature of 24 - 27 °C and a light time of 12 h / 24 h for 15 - 30 d to obtain cluster buds.

[0017] Further, the specific step S4 is as follows: Only retain the tillering tissue mass of the cluster buds in S3, immerse it in liquid medium C for 24 - 72 h, and shake it once every 1 - 2 h during the immersion period to obtain the tillering tissue mass after polyploid induction.

[0018] Further, the specific step S5 is as follows: Inoculate the tillering tissue mass after polyploid induction in S4 onto liquid medium D for culture for 20 - 40 d to obtain regenerated plants.

[0019] Further, in step S7, the time for plant acclimatization is 2 - 3 months.

[0020] Further, the composition of medium B includes MS medium, 0.5 - 2 mg / L cytokinin 6 - BA, and 0.3 - 1.3 mg / L kinetin KT.

[0021] Further, the composition of the liquid medium C includes MS medium, 2.5 - 7.5 mg / mL colchicine, and 0.3 - 1.3 mg / L kinetin KT.

[0022] Further, the composition of the liquid medium D includes MS medium, 0.5 - 2 mg / L naphthaleneacetic acid NAA, and 0.3 - 1.3 mg / L kinetin KT.

[0023] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0024] 1. By using the induction method of the present invention, on the basis of rapid propagation of Arundo donax tissue culture, the culture medium can be scientifically configured, the culture conditions can be reasonably adjusted, and the Arundo donax polyploid with excellent traits can be obtained quickly and efficiently.

[0025] 2. The present invention selects a specific concentration range of colchicine for induction, significantly improving the induction rate of Arundo donax polyploid and effectively reducing the incidence of chimeras.

[0026] 3. Traditionally, the induction materials for Arundo donax polyploid are embryogenic callus or callus. The present invention uses the tillering tissue mass of clustered buds as the induction material. It is easier to obtain materials for polyploid induction using tillering tissue. The present invention not only enriches the existing induction materials for Arundo donax polyploid, but also because there is a high division activity in the tillering tissue of Arundo donax, compared with inducing all clustered buds and callus, inducing the tillering tissue mass can significantly improve the induction rate of Arundo donax polyploid, reduce the incidence of chimeras, save time and cost, and provide a new way for the breeding of new Arundo donax varieties. Description of the Drawings

[0027] Figure 1 It is a display diagram of inoculating the pretreated explant.

[0028] Figure 2 It is a display diagram of clustered buds.

[0029] Figure 3 It is a display diagram of the Arundo donax tetraploid after colchicine induction of the tillering tissue mass. Detailed Embodiments

[0030] To better understand the technical content of the present invention, specific embodiments are provided below to further illustrate the present invention.

[0031] The experimental methods used in the embodiments of the present invention are all conventional methods unless otherwise specified.

[0032] The materials, reagents, etc. used in the embodiments of the present invention can be obtained from commercial channels unless otherwise specified.

[0033] Example 1 - Colchicine 5.0 mg / mL

[0034] A method for inducing Arundo donax polyploid, comprising the following steps:

[0035] S1. When new shoots of Arundo donax grow out, the bottom new leaves are growing but the top bracts have not fallen off and the leaves have not emerged, collect healthy stem segments of Arundo donax, remove the bracts, cut them into 2-3 cm stem segments with buds, wash them, and in a sterile environment, first soak them in a 70% v / v ethanol solution for 45 s, rinse them 2 times with sterile water, then soak them in a 0.1% w / v mercuric chloride solution for 11 min, and rinse them 6 times with sterile water to obtain pretreated explants;

[0036] S2. In a sterile environment, cut off 2-3 mm from each end of the stem segment of the pretreated explant in S1, inoculate it on an MS solid medium, with the bud point contacting the surface of the medium, and culture it for 22 d under the light condition of 25±1°C and 12 h / 24 h to obtain sterile buds;

[0037] S3. Cut the sterile buds in S2 into stem segments with buds, inoculate them on medium B, and culture them for 22 d under the light condition of 25±1°C and 12 h / 24 h to obtain cluster buds;

[0038] Wherein medium B is: MS medium + 1.2 mg / L cytokinin 6-BA + 0.8 mg / L kinetin KT;

[0039] S4. Only retain the tillering tissue masses of the cluster buds in S3, soak them in liquid medium C for 24 h, 48 h, and 72 h respectively, and shake them once every 1.5 h during the soaking period to obtain tillering tissue masses after polyploid induction;

[0040] Wherein liquid medium C is: MS medium + 5.0 mg / mL colchicine + 0.8 mg / L kinetin KT;

[0041] S5. Inoculate the tillering tissue masses after polyploid induction in S4 on liquid medium D and culture them for 30 d to obtain regenerated plants;

[0042] Wherein liquid medium D is: MS medium + 1.2 mg / L auxin NAA + 0.8 mg / L kinetin KT;

[0043] S6. Identify the ploidy of the leaves of the regenerated plants in S5 by flow cytometry;

[0044] S7. Plant the regenerated plants in S6 in a sand bed for acclimatization for 2.5 months. When the regenerated plants grow to more than 20 cm, take the leaves and conduct re-identification by flow cytometry.

[0045] Example 2 - Colchicine 2.5 mg / mL

[0046] A method for inducing Arundo donax polyploid, comprising the following steps:

[0047] S1. When the new shoots of Arundo donax grow out, the new leaves at the bottom grow, but the bracts at the top have not fallen off and the leaves have not emerged, collect healthy stem segments, remove the bracts, cut them into 2 - 3 cm stem segments with buds, scrub them with washing powder, and rinse them thoroughly with tap water. In a sterile environment, first soak them in a 70% v / v ethanol solution for 30 s, rinse them once with sterile water, then soak them in a 0.1% w / v mercuric chloride solution for 10 min, and rinse them 5 times with sterile water to obtain the pretreated explants;

[0048] S2. In a sterile environment, cut off 2 - 3 mm from each end of the stem segments of the pretreated explants in S1, inoculate them on the MS solid medium, with the bud points in contact with the surface of the medium, and culture them for 15 d under the light condition of 25 ± 1°C and 12 h / 24 h to obtain sterile buds;

[0049] S3. Cut the sterile buds in S2 into stem segments with buds, inoculate them on Medium B, and culture them for 15 d under the light condition of 25 ± 1°C and 12 h / 24 h to obtain clustered buds;

[0050] Wherein Medium B is: MS medium + 0.5 mg / L cytokinin 6 - BA + 0.3 mg / L kinetin KT;

[0051] S4. Only retain the tillering tissue masses of the clustered buds in S3, immerse them in Liquid Medium C for 24 h, 48 h, and 72 h respectively, and shake them once every 1 h during the immersion period to obtain the tillering tissue masses after polyploid induction;

[0052] Wherein Liquid Medium C is: MS medium + 2.5 mg / mL colchicine + 0.3 mg / L kinetin KT;

[0053] S5. Inoculate the tillering tissue masses after polyploid induction in S4 on Liquid Medium D and culture them for 20 d to obtain regenerated plants;

[0054] Wherein Liquid Medium D is: MS medium + 0.5 mg / L auxin NAA + 0.3 mg / L kinetin KT;

[0055] S6. Identify the ploidy of the leaves of the regenerated plants in S5 by flow cytometry;

[0056] S7. Plant the regenerated plants in S6 on a sand bed for acclimatization for 2 months. When the regenerated plants grow to more than 20 cm, take the leaves and perform re - identification by flow cytometry.

[0057] Example 3 - Colchicine 7.5 mg / mL

[0058] A method for inducing polyploid Arundo donax, comprising the following steps:

[0059] S1. When the new shoots of Arundo donax grow out, the new leaves at the bottom grow, but the bracts at the top have not fallen off and the leaves have not emerged, collect healthy stem segments, remove the bracts, cut them into 2 - 3 cm stem segments with buds, scrub them with washing powder, and rinse them thoroughly with tap water. Under a sterile environment, first soak them in a 70% v / v ethanol solution for 60 s, rinse them 3 times with sterile water, then soak them in a 0.1% w / v mercuric chloride solution for 12 min, and rinse them 7 times with sterile water to obtain the pretreated explants;

[0060] S2. Under a sterile environment, cut off 2 - 3 mm from each end of the stem segments of the pretreated explants in S1, inoculate them on the MS solid medium, with the bud points contacting the surface of the medium, and culture them for 30 d under the light condition of 25 ± 1 °C, 12 h / 24 h to obtain sterile buds;

[0061] S3. Cut the sterile buds in S2 into stem segments with buds, inoculate them on Medium B, and culture them for 30 d under the light condition of 25 ± 1 °C, 12 h / 24 h to obtain cluster buds;

[0062] Wherein Medium B is: MS medium + 2 mg / L cytokinin 6 - BA + 1.3 mg / L kinetin KT;

[0063] S4. Only retain the tillering tissue masses of the cluster buds in S3, immerse them in Liquid Medium C for 24 h, 48 h, and 72 h respectively, and shake them once every 2 h during the immersion to obtain the tillering tissue masses after polyploid induction;

[0064] Wherein Liquid Medium C is: MS medium + 7.5 mg / mL colchicine + 1.3 mg / L kinetin KT;

[0065] S5. Inoculate the tillering tissue masses after polyploid induction in S4 on Liquid Medium D and culture them for 40 d to obtain regenerated plants;

[0066] Wherein Liquid Medium D is: MS medium + 2 mg / L kinetin NAA + 1.3 mg / L kinetin KT;

[0067] S6. Identify the ploidy of the leaves of the regenerated plants in S5 by flow cytometry;

[0068] S7. Plant the regenerated plants in S6 on a sand bed for acclimatization for 3 months. When the regenerated plants grow to more than 20 cm, take the leaves and conduct re - identification by flow cytometry.

[0069] Comparative Example 1 - Colchicine 0.0 mg / mL

[0070] The difference from Example 1 is that Liquid Medium C does not contain colchicine, and the others are the same as Example 1.

[0071] An induction method for Arundo donax polyploid, comprising the following steps:

[0072] S1. When new shoots of Arundo donax grow, new leaves at the bottom grow but the top bracts do not fall off and the leaves do not emerge, collect healthy stem segments, remove the bracts, cut them into 2-3 cm stem segments with buds, scrub them with washing powder, and rinse them thoroughly with tap water. In a sterile environment, first soak them in 70% v / v ethanol solution for 45 s, rinse them twice with sterile water, then soak them in 0.1% w / v mercuric chloride solution for 11 min, and rinse them six times with sterile water to obtain pretreated explants;

[0073] S2. In a sterile environment, cut off 2-3 mm from each end of the stem segment of the pretreated explant in S1, inoculate it on the MS solid medium, make the bud point contact the surface of the medium, and culture it for 22 d under the light condition of 25±1℃, 12 h / 24 h to obtain sterile buds;

[0074] S3. Cut the sterile buds in S2 into stem segments with buds, inoculate them on medium B, and culture them for 22 d under the light condition of 25±1℃, 12 h / 24 h to obtain clustered buds; wherein medium B is: MS medium + 1.2 mg / L cytokinin 6-BA + 0.8 mg / L kinetin KT;

[0075] S4. Only retain the tillering tissue mass of the clustered buds in S3, immerse them in liquid medium C for 24 h, 48 h, and 72 h respectively, and shake them once every 1.5 h during the immersion period to obtain the tillering tissue mass after polyploid induction; wherein liquid medium C is: MS medium + 0.8 mg / L kinetin KT;

[0076] S5. Inoculate the tillering tissue mass after polyploid induction in S4 on liquid medium D and culture it for 30 d to obtain regenerated plants; wherein liquid medium D is: MS medium + 1.2 mg / L kinetin NAA + 0.8 mg / L kinetin KT;

[0077] S6. Identify the ploidy of the leaves of the regenerated plants in S5 by flow cytometry;

[0078] S7. Plant the regenerated plants in S6 on a sand bed for acclimatization for 2.5 months. When the regenerated plants grow to more than 20 cm, take the leaves and perform re-identification by flow cytometry.

[0079] Comparative Example 2 - Colchicine 8.0 mg / mL

[0080] The difference from Example 1 is that the concentration of colchicine in liquid medium C is 8.0 mg / mL, and the others are the same as in Example 1.

[0081] An induction method for Arundo donax polyploid, comprising the following steps:

[0082] S1. When the new shoots of Arundo donax are growing, the new leaves at the bottom are growing but the bracts at the top have not fallen off and the leaves have not emerged, collect healthy stem segments, remove the bracts, cut them into 2 - 3 cm stem segments with buds, scrub them with washing powder, and rinse them thoroughly with tap water. In a sterile environment, first soak them in a 70% v / v ethanol solution for 45 s, rinse them twice with sterile water, then soak them in a 0.1% w / v mercuric chloride solution for 11 min, and rinse them six times with sterile water to obtain the pretreated explants;

[0083] S2. In a sterile environment, cut off 2 - 3 mm from each end of the stem segments of the pretreated explants in S1, inoculate them on the MS solid medium, with the bud points in contact with the surface of the medium, and culture them for 22 d under the light condition of 25 ± 1°C and 12 h / 24 h to obtain sterile buds;

[0084] S3. Cut the sterile buds in S2 into stem segments with buds, inoculate them on medium B, and culture them for 22 d under the light condition of 25 ± 1°C and 12 h / 24 h to obtain cluster buds; where medium B is: MS medium + 1.2 mg / L cytokinin 6 - BA + 0.8 mg / L kinetin KT;

[0085] S4. Only retain the tillering tissue masses of the cluster buds in S3, immerse them in liquid medium C for 24 h, 48 h, and 72 h respectively, and shake them once every 1.5 h during the immersion period to obtain the tillering tissue masses after polyploid induction; where liquid medium C is: MS medium + 8.0 mg / mL colchicine + 0.8 mg / L kinetin KT;

[0086] S5. Inoculate the tillering tissue masses after polyploid induction in S4 on liquid medium D and culture them for 30 d to obtain regenerated plants; where liquid medium D is: MS medium + 1.2 mg / L kinetin NAA + 0.8 mg / L kinetin KT;

[0087] S6. Identify the ploidy of the leaves of the regenerated plants in S5 by flow cytometry;

[0088] S7. Plant the regenerated plants in S6 on a sand bed for acclimatization for 2.5 months. When the regenerated plants grow to more than 20 cm, take the leaves and conduct re - identification using flow cytometry.

[0089] Test Experiment 1 - Effects of Different Colchicine Concentrations on Inducing Arundo donax

[0090] Perform ploidy identification on the leaves of the regenerated plants obtained in Examples 1 - 3 and Comparative Examples 1 - 2 by flow cytometry. The identification results are shown in Table 1:

[0091] Table 1

[0092]

[0093]

[0094] As can be seen from Table 1, when the concentration of colchicine is 5.0 mg / ml (Example 1), the treatment time is 48 hours and the highest comprehensive tetraploid yield is 96.67%; when the concentration of colchicine is 2.5 mg / ml (Example 2), the treatment time is 24 hours and the highest comprehensive tetraploid yield is 30.00%; when the concentration of colchicine is 7.5 mg / ml (Example 3), the treatment time is 24 hours and the highest comprehensive tetraploid yield is 30.00%; when the concentration of colchicine is 2.5 mg / ml (Example 2), the treatment time is 48 hours and the highest comprehensive tetraploid yield is 40.00%. The difference between Comparative Example 1 and Example 1 is that no colchicine treatment is carried out, resulting in the inability to double chromosomes and obtain polyploids. The difference between Comparative Example 2 and Example 1 is that the concentration of colchicine is 8.0 mg / ml, resulting in a large number of regenerated plants dying and the inability to obtain homozygous polyploid plants at the same time.

[0095] Comparative Example 3

[0096] The difference from Example 1 is that when inducing polyploids, all the cluster buds are immersed in liquid medium C for 24 h, 48 h, and 72 h respectively, and the others are the same as Example 1.

[0097] A method for inducing polyploid Arundo donax, comprising the following steps:

[0098] S1. In the state where new shoots of Arundo donax grow, new leaves at the bottom grow but the top bracts do not fall off and the leaves do not emerge, collect healthy stem segments, remove the bracts, cut them into 2 - 3 cm stem segments with buds, scrub with washing powder, and rinse thoroughly with tap water. In a sterile environment, first soak in 70% v / v ethanol solution for 45 s, rinse 2 times with sterile water, then soak in 0.1% w / v mercuric chloride solution for 11 min, and rinse 6 times with sterile water to obtain pretreated explants;

[0099] S2. In a sterile environment, cut off 2 - 3 mm from each end of the stem segment of the pretreated explant in S1, inoculate on MS solid medium, with the bud point contacting the surface of the medium, and culture for 22 d under the light condition of 25 ± 1°C, 12 h / 24 h to obtain sterile buds;

[0100] S3. Cut the sterile buds in S2 into stem segments with buds, inoculate on medium B, and culture for 22 d under the light condition of 25 ± 1°C, 12 h / 24 h to obtain cluster buds; wherein medium B is: MS medium + 1.2 mg / L cytokinin 6 - BA + 0.8 mg / L kinetin KT;

[0101] S4. Immerse the clustered buds in step S3 in liquid medium C for 24 h, 48 h, and 72 h respectively, and shake once every 1.5 h during the immersion period to obtain the tillering tissue masses after polyploid induction. The liquid medium C is: MS medium + 5.0 mg / mL colchicine + 0.8 mg / L kinetin KT.

[0102] S5. Inoculate the tillering tissue masses after polyploid induction in step S4 on liquid medium D and culture for 30 d to obtain regenerated plants. The liquid medium D is: MS medium + 1.2 mg / L naphthylacetic acid NAA + 0.8 mg / L kinetin KT.

[0103] S6. Identify the ploidy of the leaves of the regenerated plants in step S5 by flow cytometry.

[0104] S7. Plant the regenerated plants in step S6 in a sand bed for acclimatization for 2.5 months. When the regenerated plants grow to more than 20 cm, take the leaves and identify them again by flow cytometry.

[0105] Comparative Example 4

[0106] The difference from Example 1 is that colchicine is used to induce callus, and the others are the same as Example 1.

[0107] A method for inducing polyploid of Arundo donax, comprising the following steps:

[0108] S1. Under the condition that new shoots of Arundo donax grow, new leaves at the bottom grow but the top bracts do not fall off and the leaves do not emerge, collect healthy stem segments, remove the bracts, cut them into 2 - 3 cm stem segments with buds, scrub with washing powder, and rinse thoroughly with tap water. In a sterile environment, first soak in 70% v / v ethanol solution for 45 s, rinse 2 times with sterile water, then soak in 0.1% w / v mercuric chloride solution for 11 min, and rinse 6 times with sterile water to obtain the pretreated explants.

[0109] S2. In a sterile environment, cut off 2 - 3 mm from each end of the stem segments of the pretreated explants in step S1, inoculate on MS solid medium, with the bud points contacting the medium surface, and culture at 25 ± 1 °C under a light condition of 12 h / 24 h for 22 d to obtain sterile buds.

[0110] S3. Cut the sterile buds in step S2 into small segments, remove the bud parts, retain the stem segment parts, inoculate on medium B, and culture at 25 ± 1 °C under a light condition of 12 h / 24 h for 22 d to obtain callus. The medium B is: MS medium + 1.2 mg / L cytokinin 6 - BA + 0.8 mg / L kinetin KT.

[0111] S4. Immerse the callus obtained in S3 in liquid medium C for 24 h, 48 h, and 72 h respectively, and shake it once every 1.5 h during the immersion period to obtain the callus after polyploid induction. The liquid medium C is: MS medium + 5.0 mg / mL colchicine + 0.8 mg / L kinetin KT;

[0112] S5. Inoculate the callus after polyploid induction in S4 on liquid medium D and culture it for 30 d to obtain regenerated plants. The liquid medium D is: MS medium + 1.2 mg / L naphthylacetic acid NAA + 0.8 mg / L kinetin KT;

[0113] S6. Identify the ploidy of the leaves of the regenerated plants in S5 by flow cytometry;

[0114] S7. Plant the regenerated plants in S6 on a sand bed for acclimatization for 2.5 months. When the regenerated plants grow to more than 20 cm, take the leaves and perform re-identification by flow cytometry.

[0115] Test Experiment 2 - Effects of 5.0 mg / mL Colchicine on Inducing Different Materials

[0116] Perform ploidy identification on the leaves of the regenerated plants obtained in Example 1 and Comparative Examples 3 - 4 by flow cytometry. The identification results are shown in Table 2:

[0117] Table 2

[0118]

[0119] It can be seen from Table 2 that the induction rate of using tillering tissue masses as induction materials in the present invention is significantly higher than that of Comparative Examples 3 - 4, indicating that tillering tissue masses are more suitable as induction materials for Arundo donax polyploids. Compared with traditional induction materials, the present invention enriches the induction materials for Arundo donax polyploids and improves the working efficiency of Arundo donax polyploid induction.

[0120] The above are only the preferred embodiments of the present invention and are not intended to limit the present invention. Any modifications, equivalent replacements, improvements, etc. made within the spirit and principle of the present invention shall be included within the protection scope of the present invention.

Claims

1. A method for inducing polyploidy of Phragmites australis, characterized in that: The following steps are involved: S1. Collecting robust stem segments of Phragmites australis, removing bracts, selecting stem segments with buds, washing and disinfecting, and obtaining pretreated explants; S2, inoculating the explants pretreated in S1 on MS solid culture medium, culturing under light, and obtaining sterile buds; S3, inoculating the sterile buds in S2 onto medium B, culturing under light, and obtaining clustered buds; The culture medium B comprises MS culture medium, 0.5-2 mg / L cytokinin 6-BA and 0.3-1.3 mg / L kinetin KT; S4, soaking the clustered buds in S3 in liquid culture medium C for 48-72 hours, shaking the buds every 1-2 hours, and obtaining the tillering tissue mass after polyploid induction; The liquid culture medium C comprises MS culture medium, 5.0-7.5 mg / mL colchicine and 0.3-1.3 mg / L kinetin KT; S5, inoculating the tillering tissue mass after polyploid induction in S4 on liquid culture medium D to obtain regenerated plants; The liquid culture medium D comprises MS culture medium, 0.5-2 mg / L kinetin NAA and 0.3-1.3 mg / L kinetin KT; S6, using flow cytometry to identify the ploidy of leaves of the regenerated plants in S5; S7. Plant the regenerated plants in S6 for seedling hardening. When the regenerated plants grow to more than 20 cm, take leaves for further identification.

2. The method for inducing polyploid Phragmites australis according to claim 1, characterized in that: The step S1 specifically comprises: when new shoots of Phragmites australis grow out and new leaves grow at the bottom but the bracts on the top have not fallen off and the leaves have not been pulled out, collecting robust stem segments of Phragmites australis, removing the bracts, cutting into 2-3 cm stem segments with buds, washing, soaking in a 70% v / v ethanol solution for 30-60 seconds in a sterile environment, rinsing with sterile water for 1-3 times, then soaking in a 0.1% w / v mercuric chloride solution for 10-12 minutes, and rinsing with sterile water for 5-7 times to obtain pretreated explants.

3. The method for inducing polyploid Phragmites australis according to claim 1, characterized in that: The step S2 specifically comprises: in a sterile environment, cutting off 2-3 mm from both ends of the explant stem segment pretreated in S1, inoculating it on MS solid culture medium, with the bud point contacting the surface of the culture medium, and culturing for 15-30 days at a temperature of 24-27° C. and a light exposure time of 12h / 24h to obtain sterile buds.

4. The method for inducing polyploid Phragmites australis according to claim 1, characterized in that: The step S3 specifically comprises: cutting the sterile buds in S2 into stem segments with buds, inoculating them on culture medium B, and culturing them for 15-30 days at a temperature of 24-27° C. and a light intensity of 12 h / 24 h to obtain clustered buds.

5. The method for inducing polyploid Phragmites australis as claimed in claim 1, characterized in that: The step S5 specifically comprises: inoculating the tillering tissue mass after polyploid induction in S4 on a liquid culture medium D and culturing for 20-40 days to obtain regenerated plants.

6. The method for inducing polyploid Phragmites australis according to claim 1, characterized in that: In step S7, the time for planting and hardening the seedlings is 2-3 months.

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